CN108699122B - Novel peptides showing hydrolytic activity and use thereof - Google Patents

Abstract

The purpose of the present invention is to provide a novel molecule that catalyzes a hydrolysis reaction and is different from an enzyme protein. The catalytic peptide of the present invention is a peptide that catalyzes a hydrolysis reaction, and is characterized by being composed of at least one peptide selected from the following (a1) to (a 4). (A1) A peptide consisting of at least one of the upstream region and the downstream region of the BoxA of the Tob/BTG protein; (A2) a peptide consisting of the partial region of (A1); (A3) a peptide having a hydrolytic activity, which comprises an amino acid sequence in which 1 or several amino acids are deleted, substituted, added and/or inserted in the amino acid sequence of (A1) or (A2); (A4) a peptide having a hydrolytic activity, which comprises an amino acid sequence having 85% or more identity to the amino acid sequence of (A1) or (A2).

Description

Novel peptides showing hydrolytic activity and use thereof

Technical Field

The present invention relates to novel peptides showing hydrolytic activity and uses thereof.

Background

In the protein decomposition using a biochemical method, an enzyme protein that catalyzes a hydrolysis reaction is generally used. However, it is known that an enzyme protein is easily affected in stability and easily denatured by conditions such as water, temperature, and acidity. There is therefore a need to provide new molecules which catalyze reactions like enzyme proteins.

Disclosure of Invention

Problems to be solved by the invention

The present invention therefore has for its object to provide new molecules, different from enzyme proteins, which catalyze hydrolysis reactions.

Means for solving the problems

The catalytic peptide of the present invention is a peptide that catalyzes a hydrolysis reaction, and is characterized by being composed of at least one peptide selected from the following (A1) to (C4).

(A1) Peptide comprising at least one of the upstream and downstream regions of BoxA of Tob/BTG protein

(A2) A peptide consisting of said partial region of (A1)

(A3) A peptide (A4) having a hydrolytic activity, which is composed of an amino acid sequence in which 1 or several amino acids are deleted, substituted, added and/or inserted in the amino acid sequence of (A1) or (A2), and which is composed of an amino acid sequence having 85% or more identity to the amino acid sequence of (A1) or (A2), and which has a hydrolytic activity

(B1) Peptides consisting of BoxB of Tob/BTG protein

(B2) A peptide consisting of said partial region of (B1)

(B3) A peptide having a hydrolytic activity, which comprises an amino acid sequence in which 1 or more amino acids are deleted, substituted, added and/or inserted in the amino acid sequence of (B1) or (B2) (B4) and which comprises an amino acid sequence having 85% or more identity to the amino acid sequence of (B1) or (B2), and which has a hydrolytic activity

(C1) Peptides composed of the C-terminal or intermediate region of the Tob/BTG protein

(C2) A peptide consisting of said partial region of (C1)

(C3) A peptide having a hydrolytic activity, which comprises an amino acid sequence in which 1 or several amino acids are deleted, substituted, added and/or inserted in the amino acid sequence of (C1) or (C2)

(C4) A peptide having a hydrolytic activity, which comprises an amino acid sequence having 85% or more identity to the amino acid sequence of (C1) or (C2)

The catalytic peptide reagents of the invention comprise a catalytic molecule characterized by being a catalytic peptide of the invention.

The method of decomposing a protein or a peptide of the present invention is a method of decomposing a protein or a peptide, and the method includes a step of treating a substrate, which is a protein or a peptide, with the catalytic peptide of the present invention.

ADVANTAGEOUS EFFECTS OF INVENTION

Hydrolysis reactions can be catalyzed by the catalytic peptides of the invention. Since the catalytic peptide of the present invention is a peptide having a small molecular weight unlike an enzyme protein, it can be suitably used in a hydrolysis reaction as a novel catalyst molecule different from the above-mentioned protein.

Drawings

Fig. 1 is a chromatogram showing the hydrolytic activity of a catalytic peptide.

FIG. 2 is a chromatogram showing the hydrolytic activity of a catalytic peptide and a fragment sequence obtained by the decomposition.

Fig. 3 is a chromatogram showing the effect of pH on the hydrolytic activity of catalytic peptides.

Fig. 4 is a chromatogram showing the hydrolytic activity of the catalytic peptide against SOD 1.

FIG. 5 shows the sequence of the fragment resulting from the catalytic peptide-induced cleavage.

Fig. 6 is a chromatogram showing the effect of buffer on the hydrolytic activity of catalytic peptides.

Fig. 7 is a chromatogram showing the effect of buffer concentration on the hydrolytic activity of catalytic peptides.

Fig. 8 is a chromatogram showing the pH optimum of the catalytic peptide.

Fig. 9 is a chromatogram in which the effect of a metal on the hydrolytic activity of a catalytic peptide is confirmed.

Fig. 10 is a chromatogram showing the hydrolytic activity of the catalytic peptide.

FIG. 11 is a chromatogram showing the hydrolytic activity of a catalytic peptide and a fragment sequence obtained by the decomposition.

Fig. 12 is a chromatogram in which the influence of the buffer on the hydrolytic activity of the catalytic peptide was confirmed.

FIG. 13 shows the fragment resulting from the catalytic peptide-induced decomposition.

FIG. 14 shows a fragment resulting from the decomposition induced by a catalytic peptide.

Fig. 15 is a table showing the presence or absence of the hydrolytic activity of various catalytic peptides.

Fig. 16 is a chromatogram showing inhibition of self-decomposition of a catalytic peptide by a protease inhibitor.

Fig. 17 is a chromatogram in which the influence of an organic solvent on the hydrolytic activity of a catalytic peptide was confirmed.

Fig. 18 is a chromatogram in which the effect of albumin on the hydrolysis activity of the catalytic peptide was confirmed.

Fig. 19 is a chromatogram showing inhibition of self-decomposition of a catalytic peptide by a protease inhibitor.

Fig. 20 is a graph showing the optimal concentration of catalytic peptides.

Fig. 21 is a chromatogram showing the hydrolytic activity of a catalytic peptide and the amino acid sequence of a fragment obtained by the decomposition.

Fig. 22 is a chromatogram showing inhibition of self-decomposition of a catalytic peptide by various protease inhibitors.

Fig. 23 is a graph showing the hydrolytic activity of catalytic peptides.

FIG. 24 is a chromatogram showing the hydrolytic activity of a catalytic peptide for solid A.beta.1-42 and a fragment obtained by the decomposition.

FIG. 25 is a chromatogram showing the hydrolytic activity of a catalytic peptide for soluble A.beta.1-42 and cleavage sites.

Fig. 26 is a chromatogram showing the self-decomposition of a catalytic peptide and the amino acid sequence of the fragment obtained by the decomposition.

Fig. 27 is a chromatogram showing self-decomposition of a catalytic peptide and an amino acid sequence of a fragment obtained by decomposition.

Fig. 28 is a chromatogram showing the hydrolytic activity of a β 11-29 by a catalytic peptide.

Fig. 29 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 30 is a chromatogram showing the hydrolytic activity of a catalytic peptide against a β.

FIG. 31 is a graph showing the relationship between the substrate concentration and the reaction rate.

Fig. 32 is a chromatogram showing the hydrolytic activity of a catalytic peptide against a β.

Fig. 33 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 34 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 35 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 36 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 37 is a chromatogram showing self-decomposition of catalytic peptides.

Fig. 38 is a chromatogram showing inhibition of self-decomposition of a catalytic peptide by a protease inhibitor.

Fig. 39 is a diagram showing cleavage points of a β by the catalytic peptide.

Fig. 40 is a graph showing the intermolecular interaction of catalytic peptides with a β.

FIG. 41 shows the NMR results of the catalytic peptide.

Fig. 42 is a schematic diagram showing a structure assumed for the catalytic peptide.

Fig. 43 is a chromatogram showing the hydrolytic activity of catalytic peptides on Tau.

Fig. 44 is a chromatogram showing the hydrolytic activity of catalytic peptides on Tau.

Fig. 45 is a chromatogram showing the hydrolytic activity of catalytic peptides on Tau.

FIG. 46 is a graph showing cleavage points of Tau by a catalytic peptide.

Fig. 47 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 48 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 49 is a chromatogram showing the hydrolytic activity of a β by a catalytic peptide.

Fig. 50 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 51 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 52 shows the reaction liquid composition.

Fig. 53 is a chromatogram showing self-decomposition of catalytic peptides.

Fig. 54 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 55 is a chromatogram showing the hydrolytic activity of a catalytic peptide against a β.

Fig. 56 is a chromatogram showing the hydrolytic activity of a catalytic peptide against a β.

Fig. 57 is a chromatogram showing inhibition of self-decomposition of a catalytic peptide by a protease inhibitor.

Fig. 58 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 59 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 60 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 61 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 62 is a chromatogram showing the hydrolytic activity of a β by a catalytic peptide.

Fig. 63 is a chromatogram showing the hydrolytic activity of a β by a catalytic peptide.

Fig. 64 is a chromatogram showing the hydrolytic activity of a β by a catalytic peptide.

Fig. 65 is a chromatogram showing the hydrolytic activity of catalytic peptides on crystallins.

Fig. 66 is a chromatogram showing the hydrolytic activity of a catalytic peptide on crystallins.

Fig. 67 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 68 is a chromatogram showing the hydrolytic activity of a β by a catalytic peptide.

Fig. 69 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Fig. 70 is a chromatogram showing the hydrolytic activity of a catalytic peptide against a β.

Fig. 71 is a chromatogram showing self-decomposition of catalytic peptides.

Fig. 72 is a chromatogram showing the hydrolytic activity of a β by the catalytic peptide.

Detailed Description

< catalytic peptide >

As described above, the catalytic peptide of the present invention is a peptide that catalyzes a hydrolysis reaction and is characterized by being composed of at least one peptide selected from the group consisting of the peptides (A1) to (C4).

The present inventors have intensively studied and found that a peptide in a region of Tob/BTG protein having an undefined function has a hydrolytic activity for catalyzing a hydrolytic reaction. The catalytic peptide of the present invention is a short-chain small molecule, and is more stable to water, temperature, acid, etc. than, for example, an enzyme protein, and thus is less likely to be denatured. Therefore, the catalytic peptide of the present invention can be handled more easily than, for example, an enzyme protein, and can be applied to various uses instead of an enzyme protein.

The length of the catalytic peptide of the present invention has a lower limit of the number of amino acid residues of, for example, 5, 7 or 9, an upper limit of the number of amino acid residues of, for example, 22, 18 or 17, and a range of 5 to 22, 5 to 18 or 5 to 17.

The catalytic peptides of the present invention will be described with reference to the peptides (A1) to (A4) first.

The peptide (A1) is a peptide consisting of at least one of the upstream and downstream regions of BoxA of the Tob/BTG protein. The peptide (a1) may be, for example, a peptide composed of BoxA and its upstream region, a peptide composed of BoxA and its downstream region, or a peptide composed of the upstream region, the BoxA and the downstream region.

In the peptide (a1), the amino acid sequence of BoxA is not particularly limited, and includes, for example, the amino acid sequence of seq id No. 1. In SEQ ID NO. 1, e.g. Xaa₁Is Y, F or H, Xaa₂Is P or S, Xaa₃Is E or D, Xaa₄Is K or C, Xaa₅Is Y, L, C or S, Xaa₆Is S or Q, Xaa₇Is G or A, Xaa₈Is F or Y, Xaa₉Is V or I, Xaa₁₀Is H or R, Xaa₁₁Is I or V.

And (4) BoxA: sequence No. 1

HW[Xaa₁][Xaa₂][Xaa₃][Xaa₄]P[Xaa₅]KG[Xaa₆][Xaa₇][Xaa₈]RC[Xaa₉][Xaa₁₀][Xaa₁₁]

As the BoxA of SEQ ID NO. 1, the following sequences can be exemplified.

[ Table 1]

BoxA	Sequence of	Serial number
			TOB1	HWYPEKPYKGSGFRCIHI
	3
		TOB2	HWYPEKPLKGSGFRCVHI	44
BTG1	HWFPEKPCKGSGYRCIRI					45
		BTG2	HWFPEKPSKGSGYRCIRI	46
BTG3	HWYPEKPSKGQAYRCIRV				47
		BTG4	HWHSDCPSKGQAFRCIRI	48

In the (A1) peptide, the number of amino acid residues in the upstream region is, for example, 1 or 2 in terms of length, 10, 8, 6 or 4 in terms of upper limit, and 1 to 10, 1 to 8, 1 to 6 or 1 to 4 in terms of length. In the (A1) peptide, the number of amino acid residues in the downstream region is, for example, 1 or 2 in terms of length, 10, 8, 6 or 4 in terms of upper limit, and 1 to 10, 1 to 8, 1 to 6 or 1 to 4 in terms of length.

The lower limit of the number of amino acid residues in the length of the (A1) peptide is, for example, 18 or 20, the upper limit thereof is, for example, 30, 26 or 22, and the range thereof is, for example, 18 to 30, 18 to 26 or 18 to 22.

Specific examples of the (a1) peptide include a peptide JAL having the amino acid sequence of seq id No. 2. In the sequence of JAL, the underlined part corresponds to BoxA of SEQ ID NO. 3. In sequence No. 2, the underlined sequence may be replaced with any of the sequences of sequence Nos. 44 to 48, for example.

JAL (Tob 1): sequence No. 2KYEGHWYPEKPYKGSGFRCIHI

The peptide of (A2) is a peptide consisting of the partial region of (A1). The number of amino acid residues in the (A2) peptide has a lower limit of, for example, 5, 7, or 9, an upper limit of, for example, 18, 17, or 16, and a range of, for example, 5 to 18, 5 to 17, or 5 to 16, with respect to the length of the peptide.

Specific examples of the (A2) peptide include, for example, BoxA consisting of the peptide of SEQ ID NO. 1.

Specific examples of the (a2) peptide include peptides having at least one amino acid sequence selected from the group consisting of seq id nos 3 to 14. Sequence No. 3 is BoxA, and sequence Nos. 4 and 10 to 14 are peptides composed of a partial region of JAL (Tob1) of sequence No. 2 as the (A1) peptide, respectively, wherein sequence Nos. 4 and 11 to 14 are peptides composed of a partial region of BoxA (TOB1) of sequence No. 3. Further, SEQ ID Nos. 5 to 9 are peptides each composed of a partial region of the BoxA of SEQ ID Nos. 1 and 3.

[ Table 2]

The peptide of (A3) is a peptide having a hydrolytic activity, which comprises an amino acid sequence in which 1 or several amino acids are deleted, substituted, added and/or inserted in the amino acid sequence of (A1) or (A2). In the above (A3), the number of amino acid residues to be deleted, substituted, added and/or inserted is not particularly limited, and is, for example, 1 to 5, 1 to 3, 1 or 2.

Specific examples of the peptide (A3) include peptides having amino acid sequences of SEQ ID Nos. 15 to 26 and SEQ ID Nos. 50 and 53.

[ Table 3]

Specific examples of the peptide (A3) include peptides having amino acid sequences of SEQ ID Nos. 27 to 31. Xaa is W, L, V, N or D in SEQ ID NO. 27, Xaa is K, V, T, Y or M in SEQ ID NO. 28, Xaa is T, E, P, W or K in SEQ ID NO. 29, Xaa is H, V, W, Y, R, L, P, M, E, A, D, Q, N, K or G in SEQ ID NO. 30, and Xaa is T, Q, V, K or E in SEQ ID NO. 31.

[ Table 4]

The peptide of (A4) is a peptide having a hydrolytic activity, which comprises an amino acid sequence having 85% or more identity to the amino acid sequence of (A1) or (A2). The consistency is, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. Identity is the degree of identity when properly aligned between the sequences to be compared and means the occurrence (%) of exact identity of amino acids between the sequences. For consistency, for example, the presence of gaps in the sequence and the nature of the amino acids need to be considered. The alignment can be performed by using any algorithm, and specifically, BLAST (Basic local alignment-ment search tool), BLAST-2, FASTA, Smith-Waterman, ALIGN, Megasalin and the like homology search software can be used. The calculation of the identity can be carried out, for example, using the known homology search programs, and for specific examples, can be calculated by using preset parameters in the homology algorithm BLAST (http:// www.ncbi.nlm.nih.gov/BLAST /) of the National Center for Biotechnology Information (NCBI). The same applies to consistency.

The catalytic peptides of the present invention will be described below with respect to the peptides (B1) to (B4).

The peptide of (B1) is a peptide consisting of BoxB of Tob/BTG protein.

In the (B1) peptide, the amino acid sequence of BoxB is not particularly limited, and examples thereof include the amino acid sequence of seq id No. 32. In SEQ ID NO. 32, for example, Xaa₁Is V or L, Xaa₂Is Q, E, S or K, Xaa₃Is D or E, Xaa₄Is L or M, Xaa₅Is S or T, Xaa₆Is V, L or I, Xaa₇Is V or I, Xaa₈Is F, Y or C, Xaa₉Is E or R, Xaa₁₀Is S or C, Xaa₁₁Is Y or C, Xaa₁₂Is Q or R, Xaa₁₃Is I or Y.

And (4) BoxB: serial number 32

[Xaa₁]P[Xaa₂][Xaa₃][Xaa₄][Xaa₅][Xaa₆]W[Xaa₇]DP[Xaa₈][Xaa₉]V[Xaa₁₀][Xaa₁₁][Xaa₁₂][Xaa₁₃]GE

Specific examples of the aforementioned BoxB of SEQ ID NO. 32 (B1) include peptides having amino acid sequences of SEQ ID NO. 33 to 35.

[ Table 5]

BoxB	Sequence of	Serial number
			BTG1	LPSELTLWVDPYEVSYRIGE	33
TOB1	LPQDLSVWIDPFEVSYQIGE					34
			BTG 3	LPKELTLWVDPCRVCCRYGE	35

The peptide of (B2) is a peptide consisting of the partial region of (B1). The lower limit of the number of amino acid residues in the length of the (B2) peptide is, for example, 5, 7 or 9, the upper limit thereof is, for example, 19, 18 or 17, and the range thereof is, for example, 5 to 19, 5 to 18 or 5 to 17.

Specific examples of the peptide (B2) include peptides having the amino acid sequence of SEQ ID Nos. 36 to 38.

[ Table 6]

The peptide of (B3) is a peptide having a hydrolytic activity, which comprises an amino acid sequence in which 1 or several amino acids are deleted, substituted, added and/or inserted in the amino acid sequence of (B1) or (B2). In the above (B3), the number of amino acid residues to be deleted, substituted, added and/or inserted is not particularly limited, and is, for example, 1 to 5, 1 to 3, 1 or 2.

The peptide of (B4) has a hydrolytic activity and is composed of an amino acid sequence having 85% or more identity to the amino acid sequence of (B1) or (B2). The consistency is, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

The catalytic peptides of the present invention will be described below with respect to the peptides (C1) to (C4).

The peptide (C1) is a peptide consisting of the C-terminal region or the middle region of the Tob/BTG protein.

Specific examples of the (C1) peptide include peptides having an amino acid sequence of any one of SEQ ID Nos. 39 to 41.

[ Table 7]

The peptide of (C2) is a peptide consisting of the partial region of (C1). The lower limit of the number of amino acid residues in the length of the (C2) peptide is, for example, 5, 7 or 9, the upper limit thereof is, for example, 23, 15 or 13, and the range thereof is, for example, 5 to 23, 5 to 15 or 5 to 13.

The peptide (C3) has a hydrolytic activity, and is composed of an amino acid sequence in which 1 or several amino acids are deleted, substituted, added, and/or inserted in the amino acid sequence (C1) or (C2). In the above (C3), the number of residues of the amino acid to be deleted, substituted, added and/or inserted is not particularly limited, and is, for example, 1 to 5, 1 to 3, 1 or 2.

Specific examples of the peptide (C3) include peptides having the amino acid sequence of SEQ ID NO. 42 or 43.

[ Table 8]

BTG 3239-252P 246A-DRNHWINAHMLAPH Serial number 42

BTG 3239-252P 246A P251A-DRNHWINAHMLAAH Serial No. 43

The peptide of (C4) is a peptide having a hydrolytic activity, which comprises an amino acid sequence having 85% or more identity to the amino acid sequence of (C1) or (C2). The consistency is, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

The catalytic peptide of the present invention can hydrolyze a protein or peptide as a substrate, for example. The substrate is not particularly limited, and examples thereof include amyloid β (a β) protein or a fragment peptide thereof, prion protein or a fragment peptide thereof, hMMP7 (human matrix metalloproteinase 7) or a fragment peptide thereof, SOD1 (superoxide dismutase 1) or a fragment peptide thereof, Tau protein such as Tau MBD or a fragment peptide thereof, and crystal protein such as α a-crystal protein or a fragment peptide thereof.

The catalytic peptide of the present invention can also be used for hydrolysis of, for example, an aggregating protein or a fragment peptide thereof. Amyloid beta, prion and SOD1 are reported to be responsible for alzheimer's disease, creutzfeldt-jakob disease (CJD) or Amyotrophic Lateral Sclerosis (ALS) due to aggregation, respectively. However, no protease has been reported which can decompose these aggregated proteins. On the one hand, the catalytic peptides of the present invention can decompose aggregated amyloid beta and prion proteins, even SOD1, for example. Therefore, it can be said that the catalytic peptide of the present invention is useful as a therapeutic agent for neurological diseases such as alzheimer's disease, creutzfeldt-jakob disease (CJD), and Amyotrophic Lateral Sclerosis (ALS) caused by the aggregated protein.

The catalytic peptides of the invention may further have properties such as autodecomposition. In this case, when the therapeutic drug (medicine) is administered to a living body, it is possible to exhibit a catalytic function and gradually decompose the drug, for example, and thus the safety is excellent.

< catalytic peptide reagent >

As previously described, the catalytic peptide reagents of the invention comprise a catalytic molecule and are characterized by the catalytic molecule being the catalytic peptide of the invention. For the purposes of the present invention, the catalytic molecule is characterized by comprising the catalytic peptide of the invention, the other structures being not subject to any restriction.

The catalytic peptide agents of the invention, for example, further comprise a molecule distinct from the catalytic peptide, which can be attached to the catalytic peptide. Such as binding molecules for targets of the dissociation object. The binding substance is, for example, a protein or a peptide, and specific examples thereof include a ligand and the like. In the catalytic peptide reagent of the present invention, it is preferable to, for example, link a binding molecule that binds to the target to the catalytic peptide for use. In this form, for example, a catalytic peptide agent of the invention is capable of binding to the target via the binding molecule and decomposing the target via the catalytic peptide of the catalytic peptide agent.

< decomposition method >

As described above, the method for degrading a protein or a peptide of the present invention includes a step of treating a substrate, which is a protein or a peptide, with the catalytic peptide of the present invention. The present invention is characterized by using the catalytic peptide, and other steps and conditions are not limited at all. Examples of the substrate include the target.

The conditions of the treatment step are not particularly limited, and the reaction temperature is, for example, room temperature to 37 ℃ and the reaction pH is, for example, 6.5 to 8. In the treatment step, albumin (albumin) may be used in combination, for example. The substrate is not particularly limited, and the above description can be continued.

Examples

[ example 1]

(1) Confirmation of hydrolytic Activity

It has been confirmed that synthetic peptide JAL has a hydrolytic activity against a synthetic peptide derived from the prodomain of hMMP7 (human matrix metalloproteinase 7).

JAL described below was used as a hydrolyzable peptide, and hMMP 742-50 or hMMP 726-50 was used as a substrate.

JAL9(Tob1)：KYEGHWYPEKPYKGSGFRCIHI

hMMP7 42-50：FYLYDSETK

hMMP7 26-50：GMSELQWEQAQDYLKRFYLYDSETK

First, JAL (final concentration 0.2mmol/L) and a fragment peptide of hMMP7 (final concentration 0.05mmol/L) were added to a buffer (Tris-HCl, pH6.5, final concentration 100mmol/L), and the reaction mixture was incubated at 37 ℃ for 14 days or 16 days. Then, the reaction mixture was subjected to HPLC to confirm the peaks of the fragment peptides of JAL and hMMP 7. HPLC was performed by the following conditions.

Column: SHISEIDO CAPCELL PAK C18 MGII (4.6mm ID. X150 mm)

Temperature: 40 deg.C

Wavelength: 220nm

Concentration gradient: 0.1% TFA 0-70% CH₃CN, 15 minutes

A detector: photodiode-array

Then, in HPLC, peaks were separated every 20. mu.L, and identification of fragments was performed using mass spectrometry (ABI QSTAR Elite system Co., Ltd.) (hereinafter referred to as MS). MS was performed using flow injection analysis and by the following conditions.

MS: positive ion mode

Ion spray voltage: 3500-5500V

Ion source temperature: 140 ℃ and 400 DEG C

Solvent: 0.1% of HCOOH 70% CH₃CN

In the following experiments, the hydrolytic activity was measured under the same conditions unless otherwise specified.

The results are shown in fig. 1 and 2. FIG. 1 shows the HPLC results 0 day and 14 days after the start of the reaction. As shown in FIG. 1, it is clear that JAL has activity on hMMP 742-50 and hMMP 726-50, as the peaks of hMMP 742-50 and hMMP 726-50 are reduced. Further, it is considered that self-decomposition occurs when the peak of JAL itself becomes low. FIG. 2 is a result of identifying fragments from MS 16 days after the start of the reaction, showing the sequence of the fragments obtained by the decomposition. As shown in FIG. 2, the substrate is cleaved at multiple sites.

(2) Study of optimum pH

The optimum pH for activity was investigated using JAL as the hydrolyzed peptide and hMMP 71-42 as the substrate. The measurement was carried out in the same manner as in (1) above, except that the pH of the reaction solution was adjusted in 0.5 scale within the range of pH3.5 to 8.0 and the reaction solution was cultured for 5 days. As for the adjustment of pH, acetic acid buffer was used in the range of pH3.5 to 6.0, and Tris-HCl buffer was used in the range of pH6.5 to 8.0, to give final concentrations of 150 mmol/L. As a result, the activity was found to be between pH3.5 and 6.0 (FIG. 3). In addition, no difference was observed between the results at pH6.5-8.0 and day 0 after the start of the reaction.

hMMP7 1-42：

MRLTVLCAVCLLPGSLALPLPQEAGGMSELQWEQAQDYLKRF (Serial number 49)

[ example 2]

(1) Confirmation of hydrolytic Activity

JAL has been confirmed to have a hydrolytic activity against a fragment peptide derived from SOD1 (superoxide dismutase 1).

The hydrolytic activity was measured in the same manner as in example 1(1) except that JAL was used as the hydrolytic peptide, the following SOD 12-38, SOD 112-38, SOD 153-70, SOD 1115-154 or SOD 12-12 was used as the substrate, the pH of the reaction solution was 6.5 or 7.5, and the incubation time of the reaction was 0 day, 3 days or 4 days.

SOD1 2-38：ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKG

SOD1 12-38：DGPVQGIINFEQKESNGPVKVWGSIKG

SOD1 53-70：DNTAGCTSAGPHFNPLSR

SOD1 115-154：GRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ

SOD1 2-12：ATKAVCVLKGD

The results are shown in fig. 4 and 5. FIG. 4 shows the results of HPLC at 0 day and 3 days or 4 days after the start of the reaction. As shown in FIG. 4, the activity of JAL on SOD 12-38, SOD 112-38, SOD 153-70 and SOD 12-12 was confirmed. Especially has strong activity on SOD 153-70 and SOD 12-38. In one aspect, no activity was observed for SOD 1115-154. FIG. 5 shows the result of fragment identification according to MS. As shown in FIG. 5, in SOD 12-38 and SOD 12-12, a plurality of sites indicated by arrows were cut by JAL. In addition, S-S binding between Cys residues was observed in the fragment peptide fragment derived from SOD 12-38.

(2) Examination of buffer solution (reaction solution)

The buffers used in the reaction solution were investigated using JAL as a hydrolyzable peptide and SOD 12-38 as a substrate. Except that Tris buffer (Tris-HCl, pH6.5, final concentration 100mmol/L), assay buffer (50mmol/L Tris-HCl, pH7.5, 150mmol/L NaCl, 10mmol/L Ca were used²⁺、5μmol/L Zn²⁺0.06% Briji35 and 0.02% NaN₃) The buffer solution was cultured for a period of time other than 0 day, 1 day, and 3 days, and the measurement was performed in the same manner as in (1). In addition, as a control group, physiological saline (0.9 w/v%) was used instead of the buffer. As a result, cleavage by JAL was observed earlier in the test buffer than in the Tris buffer (FIG. 6). In addition, no activity was observed in physiological saline. Therefore, it is considered that the use of a buffer is essential for the hydrolytic activity of JAL. Physiological saline is considered to be suitable for, for example, JAL before storage and use.

(3) Investigation of the concentration

Next, measurement was performed in the same manner as in (2) above, except that the concentration of Tris buffer was changed and the incubation time was changed to 0 day, 1 day, and 4 days. As a result, no activity was observed in MilliQ water (0mmol/L) alone, whereas activity was observed in Tris buffer independent of the concentration (FIG. 7).

(4) Study of optimum pH

Next, the optimum pH for the activity was investigated using JAL as a hydrolysis peptide and SOD 12-38 as a substrate. The measurement was carried out in the same manner as in (1) above except that the pH of the reaction solution was adjusted in 0.5 steps within the range of pH6.5 to 8.0 and the incubation time was 0 day or 1 day. As a result, activity was observed at any pH, and no difference was observed between the respective pH (FIG. 8). Therefore, it is considered that the pH is not affected by local pH fluctuation when used in vivo.

(5) Study of Metal requirement

Further, the metal-requiring property of the activity was investigated using JAL as a hydrolysis peptide and SOD 12-38 as a substrate. The metal ion added in the reaction solution is Zn²⁺、Ca²⁺、Co²⁺And combinations thereof. In each reaction solution, Ca is a concentration of the metal ion²⁺10mmol/L and 5. mu. mol/L of other metal ions. Then, the respective reaction solutions were measured in the same manner as in the above (1) except that the metal ions were not added to the reaction solutions and the incubation time was 0 day or 1 day. As a result, the catalyst was active regardless of the presence or absence of the metal, and no metal ion (Zn) was observed²⁺、Co²⁺、Ca²⁺) The presence or absence of (2) difference (FIG. 9).

[ example 3]

It was confirmed that a variant of JAL has a hydrolytic activity.

(1) Activity on SOD 12-38 and SOD 12-12

The hydrolytic activity was measured in the same manner as in example 1(1) except that JAL7-22, JAL14-22, JAL1-22C19M or JAL 7-22C19M described below was used as a hydrolytic peptide and the SOD 12-38 and SOD 12-12 described above were used as substrates as JAL variants. Since the Cys residues contained in JAL form S-S bonds during the reaction, which makes fragment analysis difficult, JAL1-22C19M and JAL 7-22C19M are obtained by replacing the Cys residues of JAL1-22 and JAL7-22 with Met. As a result, activities were observed in JAL7-22, JAL1-22C19M and JAL 7-22C19M (FIG. 10).

JAL7-22：YPEKPYKGSGFRCIHI

JAL14-22：GSGFRCIHI

JAL1-22 C19M：KYEGHWYPEKPYKGSGFRMIHI

JAL7-22 C19M：YPEKPYKGSGFRMIHI

(2) Activity against Abeta 1-20

The activity was measured in the same manner as in (1) above, except that JAL was used as the hydrolyzable peptide and the following A.beta.1-20 was used as the substrate. As a result, JAL was active against A.beta.1-20 (FIG. 11).

A beta 1-20: sequence DAEFRHDSGYEVHHQKLVFF

(3) Examination of buffer solution (reaction solution)

The buffer was investigated using JAL as the hydrolyzed peptide and A.beta.1-20 as the substrate. The measurement was performed in the same manner as in (2) above, except that PBS, Tris buffer, or phosphate buffer was used as the buffer, and the incubation time was 0 day, 3 days, or 5 days. As a result, the activity was observed in PBS (pH7.4) and Tris buffer (50mmol/L, pH7.5) (FIG. 12). On the one hand, no activity was observed in phosphate buffer. This shows that the activity of JAL can be sufficiently obtained by using PBS and Tris buffer as buffers. In the following test, the measurement was performed using PBS from the viewpoint of suitability for in vivo administration.

(4) Tob/BTG Activity Studies

The activity was measured in the same manner as in (1) above except that JAL (Tob1)7-22CM, JAL (Tob1)7-22, Tob 27-22, BTG 17-22, BTG 27-22, BTG 37-22 or BTG 47-22 was used as a hydrolysis peptide and A.beta.1-20 or A.beta.11-29 was used as a substrate. In addition, human serum albumin (HSA, Wako pure chemical industries, Ltd., final concentration 0.025%) was added to measure changes in activity.

JAL(Tob1)7-22CM：YPEKPYKGSGFRMIHI

Tob2 7-22：YPEKPLKGSGFRCVHI

BTG1 7-22：FPEKPCKGSGYRCIRI

BTG2 7-22：FPEKPSKGSGYRCIRI

BTG3 7-22：YPEKPSKGQAYRCIRV

BTG4 7-22：HSDCPSKGQAFRCIRI

The results are shown in fig. 13. In FIG. 13, 1 to 19, 1 to 18, etc. show the fragmented regions. In each variant, activity on A.beta.1-20 and A.beta.11-29 was observed. In addition, the comparative results of chromatography were that the activity was enhanced by adding HSA.

(5) Activity on fragment peptides derived from PrP (human prion protein)

Next, the activities of the respective peptides were measured using PrP 175-189 described below as a substrate. In addition, Tob 27 is used-22 or BTG 37-22 as a hydrolyzable peptide assay, in which further Cu is added to HSA²⁺Resulting in a change in activity. The hydrolytic activity was measured in the same manner as in (1). As a result, the activity of Tob2, BTG1, BTG3 and BTG4 on PrP 175-189 was observed. In addition, Tob 27-22 and BTG 37-22 even if Cu is added²⁺Also active (fig. 14). In the presence of Cu²⁺The reaction system (2) was observed to have S-S binding by the peptide derived from the fragment of PrP 175-189.

PrP 175-189：FVHDCVNITIKQHTV

(6) Activity on A beta peptide

Next, the activity was measured in the same manner as in (1) above, except that various JAL variants were used as the hydrolysis reaction peptide and A.beta.1-20 or the following A.beta.11-29 was used as the substrate (FIG. 15). As a result, JAL (Tob1)7-22C19M, JAL (Tob1)12-18, JAL (Tob1)12-18S15A, and JAL (Tob1)14-18 were active against A.beta.1-20. In addition, JAL (Tob1)7-22Y7A C19M I22A, JAL (Tob1)9-20C19M, JAL (Tob1)12-22C19M I22A, JAL (Tob1)12-20C19M, and JAL (Tob1)12-20Y12A C19M have activity on A beta 11-29.

Aβ11-29：EVHHQKLVFFAEDVGSNKG

(7) Effects on autolysis by inhibitors

Next, the mechanism of autodecomposition was investigated using various JAL variants as hydrolysis peptides. A protease inhibitor (trade name: Roche cOmplete, manufactured by Roche Co.) was added to the reaction solution of each JAL variant to carry out a reaction. After 1 tablet of the protease inhibitor was dissolved in 1mL of MilliQ water, 50. mu.L of the solution was added. The hydrolytic activity was measured in the same manner as in (1). As a result, autolysis was inhibited in all the peptides tested (FIG. 16).

[ example 4]

The reaction conditions were studied for the hydrolytic activity of JAL.

(1) Influence of organic solvent

The effect of organic solvents was investigated using JAL1-22C19M as the hydrolyzed peptide and A β 1-19 as the substrate. Except that DMSO and CH were added to the reaction mixture₃OH or CH₃CN was added so that the final concentration was 10% and the culture time was not longer than 0 day, 3 days and 5 days, and the activity was measured in the same manner as in example 1 (1). As a result, even when DMSO was added to the reaction mixture, the activity of JAL1-22C19M was observed (FIG. 17). On the other hand, CH is added₃OH and CH₃No activity was observed at CN. This indicates that by dissolving a poorly soluble peptide in DMSO and using it, it is possible to use it without affecting the decomposition activity of JAL. In the following experiments, the poorly soluble peptides were dissolved in DMSO and used.

(2) Effect of Albumin

The activity measurement was carried out in the same manner as in (1) above, except that JAL 7-22C19M was used as the hydrolyzed peptide, and bovine serum albumin (BSA, Wako pure chemical industries, Ltd., final concentration 0.025%) or HSA (Wako pure chemical industries, Ltd., final concentration 0.025%) was used as the substrate. As a result, neither HSA nor BSA was cleaved by JAL (FIG. 18). On the other hand, addition of HSA and BSA to the reaction solution increased the autodecomposition of JAL 7-22C 19M. From this fact, it is considered that the activity of JAL 7-22C19M was enhanced by the addition of HSA and BSA. Further, it is considered that serum albumin is also suitable for in vivo use in the presence of serum albumin in vivo. In the subsequent experiments, the hydrolytic activity of JAL was measured by adding HSA to the reaction solution.

(3) Effects on autolysis by inhibitors

The mechanism of autodigestion was investigated using JAL 7-22C19M as a hydrolytic peptide. Activity measurement was carried out in the same manner as in the above (1) except that HSA was added to the reaction solution of JAL 7-22C19M and that protease inhibitor (trade name: Roche cOmplete), E64, aprotinin, AEBSF, EDTA (0.4mmol/L) or pepstatin A was further added. As a result, the self-resolved fragment disappeared in the sample to which Roche cOmplete and AEBSF were added (FIG. 19).

(4) Investigation of optimum concentration

The optimum concentration of the substrate for the hydrolyzed peptide was investigated using JAL 7-22C19M as the hydrolyzed peptide and A β 1-19 as the substrate. Activity measurement was performed in the same manner as in (1) above, except that the culture time was 0 day, 3 days, and 5 days. The results are shown in fig. 20. In FIG. 20, the bars for the days show the results of the concentrations of the hydrolyzed peptides of 0, 0.05, 0.1, 0.2, 0.4, and 0.8mmol/L (mM) from the left. As shown in FIG. 20, the substrate (upper panel) decreased and the degradation product (A.beta.1-18) (lower panel) increased at a ratio of 1:1 (final peptide concentration: 0.05mmol/L) and 1:4 (final peptide concentration: 0.2mmol/L) of the substrate (A.beta.1-19) and the peptide. From this fact, it is found that JAL7-22 has a strong activity at these concentration ratios. In addition, when the concentration of the peptide is high, the activity of JAL7-22 decreases.

Aβ1-19：DAEFRHDSGYEVHHQKLVF

(5) Activity on various fragments of A.beta.

The activity was measured in the same manner as in (1) except that JAL 12-20C19M was used as the hydrolysis peptide and A.beta.11-29 was used as the substrate. As a result, strong activity was observed against A.beta.11-29 (FIG. 21).

(6) Investigation of protease species

The mechanism of autolysis was investigated using JAL 12-20C19M as a hydrolyzed peptide and A β 11-29 as a substrate. Activity was measured in the same manner as in (1) above except that a protease inhibitor (trade name: Roche cOmple or AEBSF) was added to the reaction mixture of JAL 12-20C19M and the reaction was carried out for 0 day or 1 day of culture time. As a result, AEBSF, which is a serine protease inhibitor, inhibited autolysis (FIG. 22). From this point of view, it is considered that JAL may be a serine protease type peptide.

[ example 5]

It was confirmed that a variant of JAL has a hydrolytic activity on a β.

(1) Investigation of the Activity of A.beta.

JAL14-18 (SEQ ID NO: GSGFR) was confirmed to have the same activity as JAL (example 3(6)), and therefore JAL14-18 and its variants were synthesized and used as a hydrolysis peptide to measure the activity. Either A.beta.1-20 (top panel) or A.beta.11-29 (bottom panel) was used as the substrate. The activity was measured in the same manner as in example 1 (1). As a result, GSGFR, GSGVR, GSGYR and DSGFR were found to have strong activities against A.beta.1-20, and GSGHR and GSGQR were found to have strong activities against A.beta.11-29 (FIG. 23). From this result, it was clarified that the substrate specificity varied depending on the difference of 1 residue in amino acid.

(2) Investigation of the Activity of solid (insoluble) A.beta.1-42

Activity was measured in the same manner as in (1) except that JAL 12-20C19M was used as a hydrolyzed peptide, synthetic solid (insoluble) Abeta 1-42 was used as a substrate, and the culture time was 0 day and 7 days. As a result, JAL 12-20C19M cleaved A.beta.1-42 as an insoluble solid (FIG. 24). From this result, it is presumed that decomposition of A.beta.1-42 existing in vivo as an insoluble aggregate is also possible.

(3) Investigation of the Activity of soluble A.beta.

The activity was measured in the same manner as in (1) above, except that JAL 12-20C19M was used as a hydrolysis peptide and purchased Abeta 1-42 (manufactured by peptide research) was used as a substrate. As a result, it was found that JAL 12-20C19M cleaved A.beta.1-42 (FIG. 25).

[ example 6]

(1) Self-decomposition of BoxB

Autodecomposition was confirmed in BoxB. Activity was measured in the same manner as in example (1) except that either of BTG 18-20 of BoxB or Tob 18-20 of BoxB was used as a hydrolysis peptide and HSA was added thereto, and the culture time was set to 0 day or 5 days. As a result, fragments showing self-decomposition were observed (FIG. 26).

(2) Autolysis of fragment peptides from the middle region of Tob1

The activity was measured in the same manner as in (1) except that Tob 1198-221 or Tob 1221-236 was used as the hydrolyzable peptide and the culture time was 0 day or 5 days. As a result, it was observed that the fragments shown in Tob 1221-236 are self-resolved (FIG. 27).

(3) Investigation of the Activity of A.beta.

The activity was measured in the same manner as in (1) except that Tob 1198-221, Tob 1221-236, Tob 18-20 of BoxB or BTG 18-20 of BoxB was used as the hydrolysis peptide and A.beta.11-29 was used as the substrate. As a result, an activity was observed in Tob 1221-236, BTG 18-20 of BoxB (FIG. 28).

[ example 7]

Various properties were investigated and the structure was analyzed for the partial sequence JAL-TA9 of JAL1-22C19M, which is a JAL variant.

JAL1-22 C19M：KYEGHWYPEKPYKGSGFRMIHI

JAL-TA 9: PYKGSGFRMI (Serial number 50)

Soluble powder A.beta.42 (standard sample, manufactured by peptide research), A.beta.1-20, A.beta.11-29, and soluble A.beta.28-42 as fragment peptides were used as substrates. The activity was measured in the same manner as in example 1(1) except that a reaction solution having the following reaction solution composition 1 was used, the reaction solution was incubated at 37 ℃ for a predetermined period of time, and 10. mu.L of the reaction solution was fed to HPLC. In the case of MS analysis, 20. mu.L of the reaction solution was subjected to HPLC and the peak was separated, followed by MS analysis.

Aβ1-42：

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

Aβ1-20：DAEFRHDSGYEVHHQKLVFF

Aβ11-29：EVHHQKLVFFAEDVGSNKG

Aβ28-42：KGAIIGLMVGGVVIA

[ Table 9]

Crystals of a β 42 were used as solid substrates. The activity was measured in the same manner as in example 1(1) except that a reaction solution having the reaction solution composition 2 was used, the reaction solution was incubated at 37 ℃ for a predetermined time, and 10. mu.L of the reaction solution was subjected to HPLC. In the case of MS analysis, 100. mu.L of the reaction solution was subjected to HPLC and after separation of peaks, MS analysis was performed.

(1) Fragments from A.beta. (A.beta. -F)

(1-1) Activity of JAL-TA9 on Abeta-F

These results are shown in fig. 29. Fig. 29 shows HPLC results of the reaction solution, with the left side showing the result of reaction time 0 and the right side showing the result of reaction after one night. As shown in FIG. 29, it was confirmed that JAL-TA9 decomposed any of A.beta. -F and showed activity against them.

(1-2) identification of cleavage Point for Abeta-F

These results are shown in fig. 30. FIG. 30 shows the result of identifying fragments by MS after a reaction for a predetermined period of time, and shows the sequence of fragments obtained by decomposition. In FIG. 30, "J" shows a fragment from JAL-TA9, and "A" shows a fragment from A β -F. As shown in FIG. 30, any of A β -F is cleaved at multiple sites. In particular, a β shows a strong cleavage activity in the middle region reported as the condensed nucleus.

(2) Reaction rates for Abeta 1-20 and Abeta 11-29

HPLC was carried out with the A.beta. -F concentrations in the reaction solution being 0.05, 0.2, 0.4mmol/L and the reaction time being 0, 1 hour. The Km value was then determined from the reduction rate of the peak of A.beta. -F. These results are shown in fig. 31. FIG. 31 is a graph showing the relationship between the substrate concentration and the reaction rate. As a result, it was found that A.beta.11-29 had a higher affinity for JAL-TA9 than A.beta.1-20.

(3)Aβ42

(3-1) Activity of JAL-TA9 on A.beta.42

The standard sample and the solid were used as a β 42. These results are shown in fig. 32. Fig. 32 shows HPLC results of the reaction solution, with solid a β 42 on the left and standard a β 42 on the right. As shown in fig. 32, it was clear that JAL-TA49 decomposed any of a β 42 and showed activity against them.

(3-2) identification of cleavage Point for Abeta 42

These results are shown in FIGS. 33 to 36. Fig. 33 and 34 show the results for solid a β 42, and fig. 35 and 36 show the results for soluble standard a β 42. In FIGS. 33-36, "J" shows a fragment from JAL-TA9, and "A" shows a fragment from A β 42. FIG. 33 shows the results of identification of fragments by MS after reaction of solid A.beta.42 for a prescribed time, and FIG. 34 shows the results of identification of fragments A5, A7, A8 by MS. FIG. 35 shows the results of identifying fragments by MS after reaction of soluble standard sample A.beta.42 at a predetermined time, and FIG. 36 shows the results of identifying fragments A1 and A4 by MS.

The soluble standard a β 42 showed a new peak at the 3-day reaction stage, and as shown in fig. 35 and 36, multiple sites were cleaved in the 7-day reaction. When the cleavage point was confirmed, a strong cleavage activity was shown particularly for a β in the middle region where the nuclei are reported to aggregate. Similarly, as shown in fig. 33 and 34, activity was also observed with solid a β 42.

(4) Self-decomposition of JAL-TA9

(4-1) confirmation of autolysis

The reaction was carried out in the same manner as in JAL-TA9 except that no substrate was added, and the self-decomposition was confirmed. The results are shown in fig. 37. As shown in FIG. 37, it was confirmed that JAL-TA9 is self-decomposing.

(4-2) influence on autolysis by inhibitor

In the case of JAL-TA9, the effect of the inhibitor on autodecomposition was confirmed. The activity was measured in the same manner except that a protease inhibitor was added to the reaction solution so that the final concentration was 0mmol/L or 6 mmol/L. In addition, as the protease inhibitor, in example 4(6), the serine protease inhibitor AEBSF which is shown to inhibit the autolysis of JAL 12-22C19M was used. The results are shown in fig. 38. In FIG. 38, the right graph shows the MS results of the reaction mixture to which the serine protease was added. As shown in fig. 38, autodecomposition was suppressed by adding AEBSF. From this result, JAL-TA9 was considered to be a serine protease type peptide.

Based on the results of (1) to (4), the cleavage points of a β 42 and a β -derived fragment are shown in fig. 39. As shown in fig. 39, it is found that a strong cleavage activity is exhibited in the middle region of a β 42.

(5) Molecular interaction of JAL-TA9 on Abeta 42 and Abeta-F

The intermolecular interaction of JAL-TA9 with A β 42 and A β -F was confirmed by using AFFINX QN μ (trade name, manufactured by inititun corporation). These results are shown in the graph of fig. 40. In FIG. 40, X is the result of the case where JAL-TA9 was not fixed to the platinum electrode, and Y is the result of the case where JAL-TA9 was fixed to the platinum electrode. As shown in FIG. 40, JAL-TA9 shows strong interactions for A β 11-29 and A β 42, which correlate with the activity of the cleavage.

(6) Structure analysis of JAL-TA9

NMR was performed on JAL-TA 9. The results are shown in fig. 41. In addition, the three-dimensional structure and active site of JAL-TA9 are shown in FIG. 42. JAL-TA9 is presumed to have a serine protease type steric structure because Lys H.epsilon.and Met H.gamma.and Ser H.beta.and Arg H.delta.are very compact because they are spatially close to each other. In addition, JAL-TA9 has a hydroxyl group (Ser) as the center, and has two amino groups (Gly) to form a negative oxygen ion Hole (Oxyanison Hole), and it is presumed that a basic amino acid, a hydroxyl group, and a C-terminal carbonyl group required for serine protease type activity are present at positions close to each other in a three-dimensional manner. It is understood that JAL-TA9 having such a low molecular weight breaks down oligomers of A β into the inside by adding a hydrolytic activity to the inside. In addition, the present invention is not limited to such presumptions.

[ example 8]

It was confirmed that JAL-TA9 causes the decomposition of Tau protein, which is one of causative substances of Alzheimer's disease.

(1) Activity of

A fragment (Tau MBD1-30) from Tau MBD (microtubule binding domain) was used as substrate. The activity was measured in the same manner as in example 1(1) except that a reaction solution having the following reaction solution composition 3 was used, the reaction solution was incubated at 37 ℃ for a predetermined time, and 10. mu.L of the reaction solution was subjected to HPLC. In the case of MS analysis, 20. mu.L of the reaction solution was subjected to HPLC and the peak was separated, followed by MS analysis.

Tau MBD 1-30: GSKDNIKHVPGGGSVQIVYKPVDLSKVTSK (Serial number 51)

[ Table 10]

These results are shown in fig. 43. Fig. 43 shows HPLC results of the reaction solution, with the left side showing the result of reaction time 0 and the right side showing the result of reaction after one night. As shown in FIG. 43, it was confirmed that JAL-TA9 decomposed Tau MBD1-30 and showed the activity.

(2) Identifying the cut-off point

FIGS. 44 and 45 show the results of identifying fragments by MS after 1 day and 5 days of reaction and the sequences of the fragments obtained by decomposition. In FIGS. 44 and 45, the upper graph shows the results of the reaction between JAL-TA9 and Tau MBD1-30, and the lower graph shows the results of the reaction mixture without JAL-TA9 added.

As shown in the lower graph of FIG. 44 (reaction time 1 day), it was confirmed that Tau MBD1-30 was decomposed without adding JAL-TA 9. As shown in the upper diagram of FIG. 44, by reacting JAL-TA9 with Tau MBD1-30, the same decomposed fragments (9, 11) of Tau MBD1-30 and self-decomposed fragments (1 to 5, 7, 8, 12, 13) of JAL-TA9 as those in the lower diagram were obtained, and fragments of Tau MBD1-30 (6, 10 underlined in the figure) which were cleaved by JAL-TA9 were also obtained. Further, as shown in FIG. 45 (reaction time 5 days), JAL-TA9 was reacted with Tau MBD1-30 to obtain the same decomposed fragments (12, 13) of Tau MBD1-30 and self-decomposed fragments (1 to 6, 9, 10, 14-16) of JAL-TA9 as those in the following figure, and also fragments of Tau MBD1-30 (4, 7, 8, 11, 13, 14 shown by underlining in the figure) which were cleaved by JAL-TA 9.

Based on these results, the cutting points of Tau MBD1-30 are shown in FIG. 46.

[ example 9]

A chimeric peptide of JAL-TA9 was synthesized, and the activity on A β was confirmed.

(1)Aβ11-29

A chimeric peptide was synthesized in which an a-AC peptide as an A.beta.binding site was bound to the N-terminus of JAL-TA 9. A.beta.11-29, which is a fragment peptide of A.beta.42, was used as a substrate. The activity was measured in the same manner as in example 1(1) except that a reaction solution having the following composition was used, the reaction solution was incubated at 37 ℃ for a predetermined time, and then 10. mu.L of the reaction solution was subjected to HPLC. In the case of MS analysis, 20. mu.L of the reaction solution after 6 days of reaction was subjected to HPLC and peaks were separated, followed by MS analysis.

Chimeric peptide (A beta binding site AAJAL-TA9)

FVIFLDVKHFSPEDLTVK-AA-YKGSGFRMI (SEQ ID NO: 52)

[ Table 11]

These results are shown in fig. 47. FIG. 47 shows the HPLC results of the reaction solution, with the substrate added on the left and the substrate not added on the right. As shown in fig. 47, it was clear that the chimeric peptide decomposed a β 11-29 and showed activity.

FIG. 48 shows the results of fragment identification from MS after 6 days of reaction and shows the sequence of the fragment obtained by decomposition.

(2)Aβ42

A β 42 was used as a substrate. The activity was measured in the same manner as in example 1(1) except that a reaction solution having the following composition was used, the reaction solution was incubated at 37 ℃ for a predetermined time, and then 10. mu.L of the reaction solution was subjected to HPLC. In the case of MS analysis, 20. mu.L of the reaction solution after 7 days of reaction was subjected to HPLC, and MS analysis was performed after separation of peaks.

[ Table 12]

These results are shown in fig. 49. FIG. 49 shows HPLC results of the reaction solution, wherein the left column shows the results of only the substrate, the middle column shows the results of only the chimeric peptide, and the right column shows the results of reacting the substrate with the chimeric peptide. As shown in fig. 49, it was confirmed that the chimeric peptide decomposed a β 4211-29 from the 1 st day of the reaction and showed activity.

FIGS. 50 and 51 show the sequences of fragments obtained by decomposition, as a result of identifying fragments from MS after 7 days of reaction. In fig. 50, underlined sequences are fragments of a β 42 cleaved by the chimeric peptide. The cleavage point of a β 42 by the chimeric peptide is also shown in fig. 50.

[ example 10]

Hydrolysis activity was confirmed for ANA-TA9 which is a variant of the partial sequence of BTG3 (SEQ ID NO: HWYPEKPSKGQAYRCIRV) of BoxA. The BTG3 is hereinafter referred to as BTG/ANA. Fig. 52 shows the composition of the reaction solution used. ANA-TA 9: SKGQAYRMI (Serial number 53)

(1) Self-decomposition

The reaction solution I in FIG. 52 was incubated at 37 ℃ for a predetermined period of time and the autolysis of ANA-TA9 was confirmed by HPLC. The results are shown in fig. 53. As shown in FIG. 53, ANA-TA9 shows autodecomposition. Fig. 53 also shows the sequence of fragments detected by the self-decomposition.

(2) Cleavage of Abeta-Fs

The activity on the A.beta.Fs shown in example 7 was confirmed. The reaction solution II in FIG. 52 was used as the composition of the reaction solution. The results are shown in fig. 54. In fig. 54, the first line shows the results of 0 hour reaction, the second line shows 1 day reaction, and the third line shows 5 days reaction. As shown in FIG. 54, ANA-TA9 was able to cleave either A β -Fs. In FIG. 54, the underlined sequence is a fragment of A β -Fs that was cleaved by ANA-TA 9.

(3) Cleavage of Abeta-42

The activity of A.beta.42 was confirmed in the standard sample shown in example 7. The reaction mixture composition used was reaction mixture III in FIG. 52. Fig. 55 and 56 show the results. As shown in fig. 55 and 56, ANA-TA9 can cleave a β 42. In FIG. 55, the underlined sequence is a fragment of A β 42 that was cleaved by ANA-TA 9.

(4) Effects caused by protease inhibitors

The effect of the protease inhibitor on the autolysis of ANA-TA9 was confirmed using the reaction solution IV shown in FIG. 57. The results of JAL-TA9 are also shown in FIG. 57. In fig. 57, the upper stage of the reaction system was 0 hour, and the lower stage was 6 hours. As shown in FIG. 57, ANA-TA9 and JAL-TA9 were each inhibited from autolysis by protease inhibitors.

[ example 11]

The hydrolytic activity was confirmed for ANA-YA4 (also called YRMI) which appeared due to the autodecomposition of ANA-TA 9.

ANA-SA 4: YRMI (SEQ ID NO. 54)

(1) Cleavage of Abeta 1-20

The reaction solution V in FIG. 52 was cultured at 37 ℃ for a predetermined period of time, and autodecomposition of ANA-YA4(YRMI) and cleavage of A.beta.1-20 by ANA-YA4 were confirmed. The results are shown in fig. 58. As shown in FIG. 58, ANA-SA4 spontaneously decomposed and decomposed A β 1-20.

The MS results for 1 day of reaction are shown in fig. 59. In FIG. 59, the underlined sequence is a fragment of A β 1-20 that was cleaved due to ANA-YA 4.

(2) Cleavage of Abeta 11-29 Abeta-F

The reaction solution V in FIG. 52 was cultured at 37 ℃ for a predetermined period of time, and autodecomposition of ANA-YA4(YRMI) and cleavage of A.beta.11-29 by ANA-YA4 were confirmed. The results are shown in fig. 60. As shown in FIG. 60, ANA-SA4 spontaneously decomposed and decomposed A β 11-29. In FIG. 60, the underlined sequence is the fragment of A β 11-29 that was cleaved due to ANA-YA 4.

The MS results for 1 day of reaction are shown in fig. 61. In FIG. 61, the underlined sequence is a fragment of A β 11-29 that was cleaved due to ANA-YA 4. In addition, the cut-off point of A β 11-29 caused by ANA-YA4 is shown in FIG. 61.

[ example 12]

The hydrolysis activity was confirmed for ANA-SA5 which appeared by the autodecomposition of ANA-TA 9.

ANA-SA 5: SKGQA (Serial number 55)

The reaction solution VI in FIG. 52 was cultured at 37 ℃ for a predetermined period of time to confirm the cleavage of A.beta.1-20 and A.beta.11-29 by ANA-SA 5. The results are shown in fig. 62. As shown in FIG. 62, ANA-SA4 decomposed A.beta.1-20, and the peak was reduced to 49.6% after 1 day of reaction, and substantially disappeared after 5 days. In addition, as shown in FIG. 63, 12 fragments of A.beta.1-20 were detected in the 5-day reaction. In addition, as shown in fig. 62, ANA-SA4 decomposed a β 11-29, and the peak was reduced to 72% after 1 day of reaction, and substantially disappeared after 5 days. In addition, as shown in FIG. 64, in the 5-day reaction, 13 fragments of A.beta.11-29 were detected. Fig. 63 and 64 show the cut-off points of the a β -Fs caused by ANA-SA 5.

[ example 13]

The α a-crystallins have chaperone type activity to maintain lens transparency. In addition, it has been reported that the 71-88 peptide of α a-crystallin inhibits aggregation of β amyloid, and the peptide itself aggregates to form amyloid fibrils. Thus, the degradation activity of the α A-crystallin was confirmed with respect to JAL-TA9 of example 7.

Fragment peptides of the following sequence of 71-88 of α a-crystallin (synthesized by kynan, university) were used as substrates. The activity was measured in the same manner as in example 1(1) except that a reaction solution having the following composition was used, the reaction solution was incubated at 37 ℃ for a predetermined time, and then 10. mu.L of the reaction solution was subjected to HPLC. In the case of MS analysis, 20. mu.L of the reaction solution was subjected to HPLC and the peak was separated, followed by MS analysis.

α a-crystallin: FVIFLDVKHFSPEDLTVK (Serial number 56)

[ Table 13]

The results are shown in fig. 65. As shown in FIG. 65, JAL-TA9 was able to decompose itself, and in addition, it was able to decompose α A-crystallin from 1 day of the reaction. In addition, as shown in FIG. 66, fragments of α A-crystallin cleaved by JAL-TA9 were detected. In FIG. 66, the underlined sequence is a fragment of α A-crystallin which is cleaved by JAL-TA 9. In addition, fig. 65 also shows the cutting points of α a-crystallin.

[ example 14]

JAL-TA9 is derived from the BoxA domain of Tob 1. Thus, a peptide (Tob1 BoxB 8-20: WIDPFEVSYQIGE) which is a BoxB domain (SEQ ID NO: 34) derived from Tob1 was assayed for activity. The same substrates as A.beta.1-20 and A.beta.11-29 in example 7 were used. The activity was measured in the same manner as in example 7, except that a reaction solution having the following reaction solution composition was used.

[ Table 14]

(1) Activity on Abeta-F

These results are shown in fig. 66. Fig. 66 shows HPLC results of the reaction solution. As shown in FIG. 66, it was shown that Tob1 BoxB 8-20, like the peptide from BoxA, decomposed A β -F and showed activity.

(2) Identification of cleavage points for Abeta-F

These results are shown in fig. 67 and 68. FIGS. 67 and 68 are the results of identification of fragments by MS, showing the sequence of the fragments obtained by decomposition. In each figure, the underlined sequence shows the fragment from a β -F. As shown in FIGS. 67 and 68, any of A.beta. -F was cleaved at multiple sites.

[ example 15]

Self-decomposition and A.beta. -F activity were confirmed for JAL12-17 (YKGSGF) and 12-16 (YKGSG). The activity was measured in the same manner as in example 7, except that a reaction solution having the following reaction solution composition was used. The same A.beta.11-29 as described in example 7 was used as a substrate. In the case of autolysis, the reaction was carried out in the same manner as above except that no substrate was added.

[ Table 15]

These results are shown in fig. 69 and 70. Both figures show the HPLC results of the reaction solution. As shown in the two figures, it has been confirmed that both JAL12-17 and JAL12-16 can decompose A.beta.F by themselves. In addition, the first and second substrates are,

FIG. 71 shows the sequence of the fragment obtained by the decomposition as a result of identifying the fragment by MS 3 days after the reaction. FIG. 71 shows the results of self-decomposition, and FIG. 72 shows the results of decomposition of A.beta. -F. As shown in the upper graph of FIG. 71, in JAL12-16, an increase in JAL 12-14(P2) is seen, and as shown in the lower graph of FIG. 71, in JAL12-17, an increase in JAL 13-17(P5) is seen.

FIG. 72 shows the sequence of the fragment obtained by the decomposition as a result of identifying the fragment by MS. In FIG. 72, the underlined sequence shows the fragment from A.beta. -F. As shown in FIG. 72, the same cutting points were found for A.beta.F in both JAL12-17 and JAL 12-16.

The present invention is explained above with reference to the embodiments and examples, but the present invention is not limited to the embodiments and examples. The construction and details of the invention are susceptible of various modifications within the scope of the invention, as will be appreciated by those skilled in the art.

This application claims priority based on U.S. provisional patent application US62/275,599, filed on 6/1/2016 and japanese patent application 2016-068496, filed on 30/3/2016, the disclosures of which are incorporated herein in their entirety.

Possibility of industrial utilization

The catalytic peptides of the invention are capable of catalyzing hydrolysis reactions. Since the catalytic peptide of the present invention is a peptide having a small molecular weight unlike an enzyme protein, it can be suitably used in a hydrolysis reaction as a novel catalytic molecule different from the above-mentioned protein.

Sequence listing

<110> Okinawa Institute of Science and Technology School Corporation

<120> novel peptide showing hydrolytic activity and use thereof

<130> TF15112WO

<150> US 62/275,599

<151> 2016-01-06

<150> JP 2016-068496

<151> 2016-03-30

<160> 58

<170> PatentIn version 3.5

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X is Y, F or H.

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X is P or S.

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is E or D.

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X is K or C.

<220>

<221> MISC_FEATURE

<222> (8)..(8)

<223> X is Y, L, C or S.

<220>

<221> MISC_FEATURE

<222> (11)..(11)

<223> X is S or Q.

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X is G or A.

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> X is F or Y.

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> X is V or I.

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> X is H or R.

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> X is I or V.

<400> 1

His Trp Xaa Xaa Xaa Xaa Pro Xaa Lys Gly Xaa Xaa Xaa Arg Cys Xaa

1 5 10 15

Xaa Xaa

<210> 2

<211> 22

<212> PRT

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Lys Tyr Glu Gly His Trp Tyr Pro Glu Lys Pro Tyr Lys Gly Ser Gly

1 5 10 15

Phe Arg Cys Ile His Ile

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His Trp Tyr Pro Glu Lys Pro Tyr Lys Gly Ser Gly Phe Arg Cys Ile

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Tyr Pro Glu Lys Pro Tyr Lys Gly Ser Gly Phe Arg Cys Ile His Ile

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Tyr Pro Glu Lys Pro Leu Lys Gly Ser Gly Phe Arg Cys Val His Ile

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Phe Pro Glu Lys Pro Cys Lys Gly Ser Gly Tyr Arg Cys Ile Arg Ile

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Phe Pro Glu Lys Pro Ser Lys Gly Ser Gly Tyr Arg Cys Ile Arg Ile

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His Ser Asp Cys Pro Ser Lys Gly Gln Ala Phe Arg Cys Ile Arg Ile

1 5 10 15

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Tyr Glu Gly His Trp Tyr Pro Glu Lys Pro Tyr Lys Gly Ser Gly Phe

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Arg

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Tyr Lys Gly Ser Gly Phe Arg

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Lys Gly Ser Gly Phe

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Gly Ser Gly Phe Arg

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Gly Ser Gly Phe Arg Cys Ile His Ile

1 5

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Lys Gly Ser Gly Phe Arg Met

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Lys Tyr Glu Gly His Trp Tyr Pro Glu Lys Pro Tyr Lys Gly Ser Gly

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Phe Arg Met Ile His Ile

20

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Tyr Pro Glu Lys Pro Tyr Lys Gly Ser Gly Phe Arg Met Ile His Ile

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Tyr Pro Ala Lys Pro Tyr Lys Gly Ser Gly Phe Arg Met Ile His Ile

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Tyr Trp Ala Lys Pro Tyr Lys Gly Ser Gly Phe Arg Met Ile His Ile

1 5 10 15

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Ala Pro Glu Lys Pro Tyr Lys Gly Ser Gly Phe Arg Met Ile His Ala

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Ala Pro Glu Ala Pro Tyr Lys Gly Ser Gly Phe Arg Met Ile His Ala

1 5 10 15

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Glu Lys Pro Tyr Lys Gly Ser Gly Phe Arg Met Ile

1 5 10

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Tyr Lys Gly Ser Gly Phe Arg Met Ile His Ala

1 5 10

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Tyr Lys Gly Ser Gly Phe Arg Met Ile

1 5

<210> 25

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Ala Lys Gly Ser Gly Phe Arg Met Ile

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<210> 26

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Tyr Lys Gly Ala Gly Phe Arg

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<210> 27

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<221> MISC_FEATURE

<222> (1)..(1)

<223> X is W, L, V, N or D.

<400> 27

Xaa Ser Gly Phe Arg

1 5

<210> 28

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<221> MISC_FEATURE

<222> (2)..(2)

<223> X is K V T, Y or M.

<400> 28

Gly Xaa Gly Phe Arg

1 5

<210> 29

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<223> X is T, E, P, W or K.

<400> 29

Gly Ser Xaa Phe Arg

1 5

<210> 30

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<212> PRT

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<223> Polypeptides

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<221> MISC_FEATURE

<222> (4)..(4)

<223> X is H, V, W, Y, R, L, P, M, E, A, D, Q, N, K or G.

<400> 30

Gly Ser Gly Xaa Arg

1 5

<210> 31

<211> 5

<212> PRT

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<220>

<223> polypeptide

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is T, Q, V, K or E.

<400> 31

Gly Ser Gly Phe Xaa

1 5

<210> 32

<211> 20

<212> PRT

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<220>

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<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> X is V or L.

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> X is Q, E, S or K.

<220>

<221> MISC_FEATURE

<222> (4)..(4)

<223> X is D or E.

<220>

<221> MISC_FEATURE

<222> (5)..(5)

<223> X is L or M.

<220>

<221> MISC_FEATURE

<222> (6)..(6)

<223> X is S or T.

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> X is V, L or I.

<220>

<221> MISC_FEATURE

<222> (9)..(9)

<223> X is V or I.

<220>

<221> MISC_FEATURE

<222> (12)..(12)

<223> X is F, Y or C.

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> X is E or R.

<220>

<221> MISC_FEATURE

<222> (15)..(15)

<223> X is S or C.

<220>

<221> MISC_FEATURE

<222> (16)..(16)

<223> X is Y is C.

<220>

<221> MISC_FEATURE

<222> (17)..(17)

<223> X is Q or R.

<220>

<221> MISC_FEATURE

<222> (18)..(18)

<223> X is I or Y.

<400> 32

Xaa Pro Xaa Xaa Xaa Xaa Xaa Trp Xaa Asp Pro Xaa Xaa Val Xaa Xaa

1 5 10 15

Xaa Xaa Gly Glu

20

<210> 33

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 33

Leu Pro Ser Glu Leu Thr Leu Trp Val Asp Pro Tyr Glu Val Ser Tyr

1 5 10 15

Arg Ile Gly Glu

20

<210> 34

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 34

Leu Pro Gln Asp Leu Ser Val Trp Ile Asp Pro Phe Glu Val Ser Tyr

1 5 10 15

Gln Ile Gly Glu

20

<210> 35

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 35

Leu Pro Lys Glu Leu Thr Leu Trp Val Asp Pro Cys Arg Val Cys Cys

1 5 10 15

Arg Tyr Gly Glu

20

<210> 36

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> Polypeptides

<400> 36

Trp Val Asp Pro Tyr Glu Val Ser Tyr Arg Ile Gly Glu

1 5 10

<210> 37

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

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<400> 37

Trp Ile Asp Pro Phe Glu Val Ser Tyr Gln Ile Gly Glu

1 5 10

<210> 38

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

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<400> 38

Trp Asn Asp Pro Cys Arg Val Cys Cys Arg Tyr Gly Glu

1 5 10

<210> 39

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 39

Ser Asn Lys Val Ala Arg Thr Ser Pro Ile Asn Leu Gly Leu Asn Val

1 5 10 15

Asn Asp Leu Leu Lys Gln Lys Ala

20

<210> 40

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 40

Ala Ile Ser Ser Ser Met His Ser Leu Tyr Gly Leu Gly Leu Gly Ser

1 5 10 15

<210> 41

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

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<400> 41

Asp Arg Asn His Trp Ile Asn Pro His Met Leu Ala Pro His

1 5 10

<210> 42

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 42

Asp Arg Asn His Trp Ile Asn Ala His Met Leu Ala Pro His

1 5 10

<210> 43

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 43

Asp Arg Asn His Trp Ile Asn Ala His Met Leu Ala Ala His

1 5 10

<210> 44

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 44

His Trp Tyr Pro Glu Lys Pro Leu Lys Gly Ser Gly Phe Arg Cys Val

1 5 10 15

His Ile

<210> 45

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 45

His Trp Phe Pro Glu Lys Pro Cys Lys Gly Ser Gly Tyr Arg Cys Ile

1 5 10 15

Arg Ile

<210> 46

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 46

His Trp Phe Pro Glu Lys Pro Ser Lys Gly Ser Gly Tyr Arg Cys Ile

1 5 10 15

Arg Ile

<210> 47

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 47

His Trp Tyr Pro Glu Lys Pro Ser Lys Gly Gln Ala Tyr Arg Cys Ile

1 5 10 15

Arg Val

<210> 48

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 48

His Trp His Ser Asp Cys Pro Ser Lys Gly Gln Ala Phe Arg Cys Ile

1 5 10 15

Arg Ile

<210> 49

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 49

Met Arg Leu Thr Val Leu Cys Ala Val Cys Leu Leu Pro Gly Ser Leu

1 5 10 15

Ala Leu Pro Leu Pro Gln Glu Ala Gly Gly Met Ser Glu Leu Gln Trp

20 25 30

Glu Gln Ala Gln Asp Tyr Leu Lys Arg Phe

35 40

<210> 50

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 50

Pro Tyr Lys Gly Ser Gly Phe Arg Met Ile

1 5 10

<210> 51

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 51

Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val Gln

1 5 10 15

Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys

20 25 30

<210> 52

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 52

Phe Val Ile Phe Leu Asp Val Lys His Phe Ser Pro Glu Asp Leu Thr

1 5 10 15

Val Lys Ala Ala Tyr Lys Gly Ser Gly Phe Arg Met Ile

20 25

<210> 53

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 53

Ser Lys Gly Gln Ala Tyr Arg Met Ile

1 5

<210> 54

<211> 4

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 54

Tyr Arg Met Ile

1

<210> 55

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 55

Ser Lys Gly Gln Ala

1 5

<210> 56

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Polypeptides

<400> 56

Phe Val Ile Phe Leu Asp Val Lys His Phe Ser Pro Glu Asp Leu Thr

1 5 10 15

Val Lys

<210> 57

<211> 6

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<400> 57

Tyr Lys Gly Ser Gly Phe

1 5

<210> 58

<211> 5

<212> PRT

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<220>

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<400> 58

Tyr Lys Gly Ser Gly

1 5

Claims (9)

1. Use of a catalytic peptide for catalyzing a hydrolysis reaction, wherein the catalytic peptide is composed of at least one peptide selected from the group consisting of the following (a1) to (C1):

(A1) a peptide consisting of at least one of the upstream region and the downstream region of BoxA of Tob/BTG protein, wherein (A1) is a peptide consisting of the amino acid sequence of SEQ ID NO. 2;

(A2) a peptide consisting of the partial region of (A1), wherein (A2) is a peptide consisting of any one of the amino acid sequences of SEQ ID NOS 4-9, 11, 13, 57 and 58;

(A3) a peptide having a hydrolytic activity, which comprises an amino acid sequence in which 1 or several amino acids are deleted, substituted, added and/or inserted in the amino acid sequence of (a1) or (a2), wherein (A3) is a peptide comprising the following amino acid sequence:

i) any one of amino acid sequences of SEQ ID NOs 16, 17, 20, 22-26, 50, 53, 54 and 55; or

ii) any one of the amino acid sequences of SEQ ID NO 27-31,

in SEQ ID NO. 27, Xaa is W, L, V, N or D,

in SEQ ID NO. 28, Xaa is K, V, Y or M,

in SEQ ID NO. 29, Xaa is T, E, P or K,

in SEQ ID NO:30 Xaa is H, V, Y, Q, N, K or G, and

in SEQ ID NO. 31, Xaa is T, Q, V, K or E;

(B2) a peptide consisting of a partial region of BoxB of Tob/BTG protein, wherein said (B2) is a peptide consisting of an amino acid sequence of any one of SEQ ID NOS: 36 or 37;

(C1) a peptide consisting of the C-terminal region or the middle region of Tob/BTG protein, wherein said (C1) is a peptide consisting of the amino acid sequence of SEQ ID NO: 40.

2. The use according to claim 1, wherein the number of amino acid residues in the peptide is in the range of 5 to 22.

3. A catalytic peptide agent comprising a catalytic molecule, wherein the catalytic molecule is a catalytic peptide;

wherein the catalytic peptide is composed of at least one peptide selected from the following (A1) to (C1):

(A1) a peptide consisting of at least one of the upstream and downstream regions of BoxA of Tob/BTG protein, wherein (A1) is a peptide consisting of the amino acid sequence of SEQ ID NO. 2;

(A2) a peptide consisting of the partial region of (A1), wherein (A2) is a peptide consisting of any one of the amino acid sequences of SEQ ID NOS 4-9, 11, 13, 57 and 58;

(A3) a peptide having a hydrolytic activity, which comprises an amino acid sequence in which 1 or several amino acids are deleted, substituted, added and/or inserted in the amino acid sequence of (a1) or (a2), wherein (A3) is a peptide comprising the following amino acid sequence:

i) any one of amino acid sequences of SEQ ID NOs 16, 17, 20, 22-26, 50, 53, 54 and 55; or

ii) any one of the amino acid sequences of SEQ ID NO 27-31,

in SEQ ID NO. 27, Xaa is W, L, V, N or D,

in SEQ ID NO. 28, Xaa is K, V, Y or M,

in SEQ ID NO. 29, Xaa is T, E, P or K,

in SEQ ID NO:30 Xaa is H, V, Y, Q, N, K or G, and

in SEQ ID NO. 31, Xaa is T, Q, V, K or E;

(B2) a peptide consisting of a partial region of BoxB of Tob/BTG protein, wherein said (B2) is a peptide consisting of an amino acid sequence of any one of SEQ ID NOS: 36 or 37;

(C1) a peptide consisting of the C-terminal region or the middle region of Tob/BTG protein, wherein said (C1) is a peptide consisting of the amino acid sequence of SEQ ID NO: 40.

4. The catalytic peptide agent of claim 3, further comprising a molecule different from the catalytic peptide, the molecule being attached to the catalytic peptide.

5. The catalytic peptide agent of claim 4, wherein the different molecule is a binding molecule to the target.

6. The catalytic peptide agent of claim 5, wherein the binding substance is a protein or a peptide.

7. A method for decomposing a protein or a peptide, comprising a step of treating a substrate with a catalytic peptide, wherein the substrate is a protein or a peptide;

wherein the catalytic peptide is composed of at least one peptide selected from the following (A1) to (C1):

(A1) a peptide consisting of at least one of the upstream region and the downstream region of BoxA of Tob/BTG protein, wherein (A1) is a peptide consisting of the amino acid sequence of SEQ ID NO. 2;

(A2) a peptide consisting of the partial region of (A1), wherein (A2) is a peptide consisting of any one of the amino acid sequences of SEQ ID NOS 4-9, 11, 13, 57 and 58;

(A3) a peptide having a hydrolytic activity, which comprises an amino acid sequence in which 1 or several amino acids are deleted, substituted, added and/or inserted in the amino acid sequence of (a1) or (a2), wherein (A3) is a peptide comprising the following amino acid sequence:

i) 16, 17, 20, 22-26, 50, 53, 54 and 55 of SEQ ID NO; or

ii) any one of the amino acid sequences of SEQ ID NO 27-31,

in SEQ ID NO. 27, Xaa is W, L, V, N or D,

in SEQ ID NO. 28, Xaa is K, V, Y or M,

in SEQ ID NO. 29, Xaa is T, E, P or K,

in SEQ ID NO:30 Xaa is H, V, Y, Q, N, K or G, and

in SEQ ID NO. 31, Xaa is T, Q, V, K or E;

(B2) a peptide consisting of a partial region of BoxB of Tob/BTG protein, wherein said (B2) is a peptide consisting of an amino acid sequence of any one of SEQ ID NOS: 36 or 37;

(C1) a peptide consisting of the C-terminal region or the middle region of Tob/BTG protein, wherein said (C1) is a peptide consisting of the amino acid sequence of SEQ ID NO: 40.

8. The decomposition method according to claim 7, wherein the substrate is an enzyme protein.

9. The degradation method according to claim 7, wherein the substrate is amyloid β or a fragment peptide thereof, prion or a fragment peptide thereof, hMMP7 or a fragment peptide thereof, SOD1 or a fragment peptide thereof, Tau protein or a fragment peptide thereof, or crystallin or a fragment peptide thereof.

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